PROJECT SUMMARY Hijacking of normal developmental programs is a common mechanism of tumorigenesis and epigenetic deregulation of developmental transcription programs is central to the genesis of most, if not all, pediatric cancers. Ewing sarcomas, aggressive bone and soft tissue tumors that predominately arise in adolescence, continue to be associated with high rates of mortality and novel approaches to therapy are needed. Ewing sarcomas are characterized by the presence of pathognomonic driver fusion oncogenes, most commonly EWS/FLI1, and their likely cellular origin is mesenchymal stem/progenitor cells (MSC). EWS/FLI1 initiates tumorigenesis by inducing widespread disruption of transcriptional regulation as a consequence of altered recruitment of chromatin remodelers to target gene enhancers and promoters. We have reported that posterior HOXD genes, in particular HOXD13, are aberrantly over-expressed by Ewing sarcoma and that posterior HOXD genes contribute to maintenance of the tumorigenic phenotype. Our preliminary studies suggest that EWS/FLI1 can induce expression of HOXD13, in a cell context dependent fashion, and that this activation is mediated by aberrant activation of developmental enhancers that are otherwise active in only very discrete spatiotemporal developmental windows. In addition, chromatin immunoprecipitation (ChIP) studies have demonstrated that in EWS/FLI1+ cells the HOXD13 promoter is preferentially enriched with the MLL- dependent activating histone modification H3K4me3 and bound by both MLL and menin proteins. Significantly, exposure of Ewing sarcoma cells to novel small molecule inhibitors of MLL:menin interaction, that are currently in preclinical development for MLL-fusion positive leukemias, leads to a dramatic loss tumorigenicity and concomitant loss posterior HOXD gene expression. Thus, these studies demonstrate that that, like MLL-fusion positive leukemias, maintenance of the oncogenic phenotype of Ewing sarcoma is critically dependent on MLL:menin-dependent activation of developmental HOX genes. In this proposal, we will test the hypothesis that HOXD13 functions an obligate cooperative oncogene in Ewing sarcoma and that dependency on its continued expression presents a previously unrecognized tumor-specific vulnerability that can be therapeutically exploited. In Aim 1 will use innovative Bru-seq technologies and functional validation studies to define the downstream transcriptional targets of HOXD13 that promote tumorigenicity. In Aim 2 we will use targeted ChIP assays and chromatin conformation studies to determine how EWS/FLI1 leads to epigenetic activation of HOXD13. In Aim 3 we will test the therapeutic potential of MI-503, a small molecule inhibitor of MLL:menin protein:protein interaction, in in vivo tumor models. These studies will together elucidate the contribution of HOXD13 to Ewing sarcoma pathogenesis and test the potential of a new class of epigenetic modifying agents for Ewing sarcoma-targeted therapies.